Association of UGT1A1 gene variants, expression levels, and enzyme concentrations with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin

Among the congener of dioxin, 2,3,7,8-TCDD is the most toxic, having a serious long-term impact on the environment and human health. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in the detoxification and excretion of endogenous and exogenous lipophilic compounds, primarily in the liver and gastrointestinal tract. This study aimed to investigate the association of UGT1A1 gene polymorphisms, expression levels, and enzyme concentration with Agent Orange/Dioxin exposure. The study included 100 individuals exposed to Agent Orange/Dioxin nearby Da Nang and Bien Hoa airports in Vietnam and 100 healthy controls. UGT1A1 SNP rs10929303, rs1042640 and rs8330 were determined by Sanger sequencing, mRNA expression was quantified by RT-qPCR and plasma UGT1A1 concentrations were measured by ELISA. The results showed that UGT1A1 polymorphisms at SNPs rs10929303, rs1042640 and rs8330 were associated with Agent Orange/Dioxin exposure (OR = 0.55, P = 0.018; OR = 0.55, P = 0.018 and OR = 0.57, P = 0.026, respectively). UGT1A1 mRNA expression levels and enzyme concentration were significantly elevated in individuals exposed to Agent Orange/Dioxin compared to controls (P < 0.0001). Benchmark dose (BMD) analyses showed that chronic exposure to 2,3,7,8-TCDD contamination affects the UGT1A1 mRNA and protein levels. Furthermore, UGT1A1 polymorphisms affected gene expression and enzyme concentrations in individuals exposed to Agent Orange/Dioxin. In conclusion, UGT1A1 gene polymorphisms, UGT1A gene expression levels and UGT1A1 enzyme concentrations were associated with Agent Orange/Dioxin exposure. The metabolism of 2,3,7,8-TCDD may influence UGT1A gene expression and enzyme concentrations.


Study areas
Bien Hoa and Da Nang Air Force Bases are considered to be major hotspots of dioxin contamination in Vietnam due to the large quantities of herbicides that were stockpiled in these areas during the war 3 .The highest concentration of dioxins ever measured at Da Nang Airport was 365,000 pg Toxicity Equivalent (TEQ)/g in soil samples collected from the former Agent Orange mixing and loading area 25 .A recent study has shown that the Toxic Equivalent Quotients (TEQs) for PCDD/F in soil and sediment samples from these areas ranged from 7.6 to 962,000 and 17 to 4860 pg/g dry weight, respectively 4 .The people who live near these areas face a high risk of dioxin exposure due to their proximity to areas heavily contaminated with Agent Orange/dioxin.Additionally, large amounts of 2,3,7,8-TCDD have been detected not only in individuals working at these airbases but also in nearby residents 5,7 .Therefore, we chose these two areas for our study.

Study subjects
The study subjects consisted of 100 participants who had been exposed to Agent Orange/dioxin (dioxin group) and 100 healthy people (control group).All subjects are part of the same Vietnamese population, specifically the Kinh ethnicity with the same genetic background.These study subjects have been used and mentioned in a previous study 26 .Briefly, dioxin group selection criteria include: (i) live near an airport for at least five years; (ii) be 18 years or older and willing to participate in the study; and (iii) have a dioxin TEQ higher than 10 pg/g lipid in their serum and a 2,3,7,8-TCDD TEQ rate in total dioxin higher than 30 percent.The inclusion criteria aimed to select subjects who were contaminated with PCDDs and PCDFs used during the war.Age, gender, and the length of time spent in dioxin-contaminated areas at the time of recruitment were collected.
The control individuals who had no exposure to Agent Orange/dioxin were recruited between February and April 2021, at Vietnam Military Medical University.The inclusion criteria for control groups include: (i) willing to take part in the study; (ii) born and raised in Northern Vietnam; (iii) not part of the army during the war in South Vietnam; and (iv) not having metabolic disorders related to the UGT1A1 enzyme, such as cancer or hyperbilirubinemia.In addition, the age of the control group was similar to that of the dioxin group.The control group was carefully selected to ensure similarity in age with the dioxin group.The control individuals underwent comprehensive clinical examinations, including abdominal ultrasound, as well as tests for blood counts and various biochemical indicators.These biochemical indicators encompassed liver enzymes such as AST, ALT, and GGT, as well as levels of glucose, urea, creatinine, and bilirubin.Furthermore, markers for hepatitis B virus (HBV) infection were also assessed.Individuals presenting with anemia, elevated liver enzyme levels, hyperbilirubinemia, and specific diseases such as diabetes, HBV infection, and cancers were excluded from the study.

Ethical approval
All study participants received thorough information about the investigation and informed consent was obtained from all participants.All methods were carried out in accordance with relevant guidelines and regulations such as clinical examination, sample and data collection and experimental procedures.The Vietnam Military Medical University Ethics Board evaluated and approved a procedure that was followed for all clinical procedures.

Quantification of UGT1A1 gene expression levels
Total RNA was extracted from whole blood using the GeneJET RNA Purification Kit (Catalog Number: K0732, Thermo Fisher Scientific, USA).Reverse transcription into cDNA was performed using the RevertAid First Strand cDNA Synthesis Kit (Catalog Number: K1622), with cycling at 65 °C for 5 min, 42 °C for 60 min, and 70 °C for 5 min, followed by storage at − 80 °C until analysis.The GAPDH gene was used as an internal control 28 .The primer sequences used for UGT1A1 gene expression were as follows: UGT1A1 forward primer 5′-ATG CTG TGG AGT CCC AGG GC-3′ and UGT1A1 reverse primer 5′-CCA TTG ATC CCA AAG AGA AAA CC-3′; GAPDH forward primer 5′-GGT GGT CTC CTC TGA CTT CAA C-3′ and GAPDH reverse primer 5′-TCT CTC TTC CTC TTG TGT TCT TG-3′ 29 .RT-qPCR was performed in a total volume of 20 µL, including 10 µL of SYBR Green PCR Master Mix 2X (Thermo Fisher Scientific, USA), 0.25 µM of each primer, 20 ng/µL of cDNA, and 6.5 µL of deionized water.A no-template control (H 2 O) was included in each reaction run.RT-qPCR for the UGT1A1 gene was performed under the following conditions: an initial denaturation step of 10 min and 20 s at 95 °C, followed by 45 cycles of 95 °C for 20 s, 60 °C for 45 s, and final extension at 72 °C for 30 s. RT-qPCR for the GAPDH gene was then performed under the following conditions: 1 cycle at 95 °C for 5 min, followed by 45 cycles of 95 °C for 30 s, 63 °C for 45 s, and final extension at 72 °C for 30 s.The expression of the UGT1A1 gene was calculated using the 2 −ΔCt method, normalizing with the GAPDH gene 30 .

Genotyping of UGT1A1 single nucleotide polymorphisms
Total DNA was extracted from whole blood samples using the Gene JET Whole Blood Genomic DNA Purification Mini Kit (ThermoFisher, USA; catalog number: K0782) following the manufacturer's instructions.A DNA fragment covering SNPs rs10929303 (1813C > T), rs1042640 (1941C > G) and rs8330 (2042C > G) of the UGT1A1 gene in Exon5N2 region was amplified by PCR reaction using previously published primer pairs 31 , as follows: UGT1A1 forward primer: 5′-AAT TAA TCA GCC CCA GAG TGC-3′ and UGT1A1 reverse primer: 5′-GAA GGC GTG TGT GTG TGA AC-3′.The temperature cycling was as follows: 95 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 54 °C for 30 s, and extension at 68 °C for 30 s, with a final extension for 5 min at 68 °C.The PCR products were purified using the GeneJet Genomic DNA Purification Kit (ThermoFisher, USA) and checked by agarose gel electrophoresis before subjecting to Sanger sequencing.The genotypes of UGT1A1 SNPs rs10929303, rs1042640, and rs8330 in the target sequences were determined using the Bioedit software and deviations from Hardy-Weinberg (HW) equilibrium were tested.UGT1A1 haplotypes were reconstructed based on three study SNPs using the expectation-maximum (EM) algorithm implemented in the Arlequin v. 3.5.1.2software.

Statistical analysis
The concentrations of 2,3,7,8-TCDD and UGT1A1 enzyme, and UGT1A1 mRNA expression levels were tested for normality and presented as mean with standard deviation or medians with ranges.These data were also logarithmic transformed for further analyses.To compare the means or medians of continuous variables, Student's t-test, ANOVA, Mann-Whitney U, or Kruskal-Wallis tests were used as appropriate.A linear regression model (univariate and multivariate) was employed to examine the correlation between variables.Particularly, the Benchmark dose (BMD) model was applied to evaluate the impact of chronic exposure to 2,3,7,8-TCDD contamination on the UGT1A1 mRNA expression and UGT1A1 protein concentrations using PROAST version 70.1.We used the Akaike information criterion (AIC) model selection to select the best-fitted models for describing the relationship between variables.The genotype and allele frequencies, as well as the haplotype of the UGT1A1, were compared between groups using Chi-square or Fisher's exact tests.Odds ratios (ORs) and their 95% confidence intervals (CIs) were determined.Arlequin 3.1 and SPSS v.25 (SPSS Statistics, IBM, Armonk, NY, USA) were used for all statistical analyses.Statistical significance was defined as two-sided P values below 0.05.

The characteristics and dioxin levels of study subjects
There was no significant difference between the mean age of the individuals exposed to Agent Orange/Dioxin (mean: 53.6 ± 10.1) and that of the healthy controls (mean: 53.0 ± 13.1).The median of blood 2,3,7,8-TCDD concentration was 43.74 pg/g lipid.The exposure time to Agent Orange/dioxin was 33 years 26 .We observed a positive correlation between the blood concentrations of 2,3,7,8-TCDD in individuals exposed to Agent Orange/ Dioxin and their duration of exposure to Agent Orange/Dioxin (r = 0.436, P < 0.0001) (Suppl.Figure 1).However, the 2,3,7,8-TCDD concentrations were not determined for the control group.

Associations of UGT1A1 gene polymorphisms with Agent Orange/Dioxin exposure
The distribution of allele and genotype frequencies of UGT1A1 gene polymorphisms rs10929303, rs1042640, and rs8330 are presented in Table 1.The results indicate that the genotype frequencies of UGT1A1 gene polymorphisms rs10929303, rs1042640, and rs8330 in the dioxin and control groups were in Hardy-Weinberg equilibrium (P > 0.05).The frequencies of minor alleles rs10929303T, rs1042640G, and rs8330G were significantly lower in individuals exposed to Agent Orange/Dioxin compared to healthy controls, suggesting that these alleles were associated with protection from Dioxin exposure (rs10929303T: OR = 0.55, 95%CI = 0.33-0.91,P = 0.018; rs1042640G: OR = 0.55, 95%CI = 0.33-0.91,P = 0.018; rs8330G: OR = 0.57, 95%CI = 0.35-0.94,P = 0.026) (Table 1).We also reconstructed UGT1A1 haplotypes based on the three study SNPs, observing two major haplotypes: CCC and TGG .The frequency of haplotype TGG was significantly lower in individuals exposed to Agent Orange/ Dioxin than in the control group, indicating an association with protection from Dioxin exposure (OR = 0.54, 95%CI = 0.32-0.89,P = 0.014).Additionally, we observed two minor haplotypes, CGG and TCG , only in individuals exposed to Agent Orange/Dioxin (Table 2).The UGT1A1 mRNA expression levels in the blood samples of the study participants were analyzed and compared between individuals exposed to Agent Orange/Dioxin and healthy controls (Fig. 1A).The results indicated a significant increase in UGT1A1 mRNA expression levels in individuals exposed to Agent Orange/Dioxin compared to healthy controls (P < 0.0001).A positive correlation was observed between UGT1A1 mRNA expression levels and 2,3,7,8-TCDD concentrations in Agent Orange/Dioxin-exposed individuals (Spearman's rho = 0.405, P < 0.0001).The benchmark dose (BMD) analyses showed that chronic exposure to 2,3,7,8-TCDD contamination affects the UGT1A1 mRNA expression levels (Fig. 1B).We incorporated the UGT1A1 mRNA expression levels and UGT1A1 enzyme concentrations into a linear regression model to examine their correlation with the concentrations of 2,3,7,8-TCDD in individuals exposed to Agent Orange/Dioxin.The findings revealed a positive correlation between UGT1A1 mRNA expression levels and 2,3,7,8-TCDD concentrations (β = 1.204;P = 0.019).

UGT1A1 gene polymorphisms and the mRNA expression
We analyzed the association between UGT1A1 gene polymorphisms and its mRNA expression in Agent Orange/ Dioxin-exposed individuals and healthy controls.In the dioxin group, individuals with genotypes rs10929303TT, rs1042640GG, and rs8330GG had the highest UGT1A1 mRNA expression levels, followed by those with genotypes rs10929303CT, rs1042640CG, and rs8330CG, and those with genotypes rs10929303CC, rs1042640CC, and rs8330CC, respectively (P < 0.0001).Individuals with the TGG haplotype had significantly higher mRNA expression compared to those with the CCC haplotype (P = 0.007).Furthermore, we observed a similar trend in the control group, but the difference did not reach statistical significance (P > 0.05) (Fig. 4 and Supp.Table 1).These results indicate that the three SNPs, rs10929303, rs1042640, and rs8330, have an effect on UGT1A1 gene expression, and alleles rs10929303T, rs1042640G, and rs8330G contribute to increased UGT1A1 gene expression.

UGT1A1 gene polymorphisms and enzyme concentrations
We hypothesized that UGT1A1 gene polymorphisms could influence plasma UGT1A1 enzyme concentrations.To examine this, we compared plasma UGT1A1 enzyme concentrations among individuals with different genotypes in two study groups.Among individuals exposed to Agent Orange/Dioxin, our results showed that individuals with genotypes rs10929303TT, rs1042640GG, and rs8330GG had the highest UGT1A1 enzyme concentrations, followed by those with genotypes rs10929303CC, rs1042640CC, and rs8330CC, and those with genotypes rs10929303CT, rs1042640CG, and rs8330CG, respectively (P < 0.0001).However, the difference in UGT1A1 enzyme concentrations among individuals with various genotypes was not significant in the control group (P > 0.05).Similarly, UGT1A1 enzyme concentrations were also not significantly different between individuals with haplotypes CCC and TGG in both the dioxin and control groups (P > 0.05) (Fig. 5 and Supp.table 1).These results suggest that UGT1A1 gene polymorphisms may affect plasma UGT1A1 enzyme concentrations in individuals exposed to Agent Orange/Dioxin but not in healthy controls.

UGT1A1 mRNA expression and UGT1A1 enzyme concentration
We performed a linear regression analysis to investigate the association between UGT1A1 mRNA expression levels and UGT1A1 enzyme concentration.The results revealed an inverse correlation between UGT1A1 mRNA expression levels and UGT1A1 enzyme concentration (Spearman's rho = − 0.313, P < 0.001) in individuals exposed to Agent Orange/Dioxin.In contrast, a positive correlation was observed between UGT1A1 mRNA expression levels and UGT1A1 enzyme concentrations in healthy controls (Spearman's rho = 0.225, P = 0.024) (Fig. 6).These findings suggest that dioxin exposure has an impact on the plasma UGT1A1 enzyme concentrations in Agent Orange/Dioxin-exposed individuals.

Discussion
In humans, TCDD is metabolized slowly and 2,3,7,8-TCDD is oxidized, chlorinated, and reduced by phase I metabolic enzymes, followed by a combined process by phase II enzymes, including the UGT1A1 enzyme 19,32 .UGT1A1 gene polymorphisms are known to affect the body's response to xenobiotic exposure such as dioxin and drugs.To our knowledge, this is the first study to show the association between UGT1A1 genetic variants and the status of exposure to Agent Orange/Dioxin containing 2,3,7,8-TCDD in the hotspots of dioxin contamination in Vietnam.The current study revealed that UGT1A1 polymorphisms at SNPs rs10929303, rs1042640 and rs8330 were associated with the status of exposure to Agent Orange/Dioxin.UGT1A1 mRNA expression levels and enzyme concentration were affected and correlated with 2,3,7,8-TCDD concentrations.The results also demonstrate the influence of UGT1A1 polymorphisms on UGT1A1 gene expression and enzyme concentrations in individuals exposed to Agent Orange/Dioxin.The distribution of UGT1A1 SNPs rs10929303 (1813C > T), rs1042640 (1941C > G) and rs8330 (2042C > G) genotypes and alleles in the Vietnamese population were similar to those reported in previous studies on Pakistani 11 and Caucasian populations 33,34 .The UGT1A1 homozygous variant (211G > A), but not the variants 1941C > G (rs1042640) and 2042C > G (rs8330), has been shown to be associated with increased bilirubin in Han Chines unconjugated hyperbilirubinemia neonates 35,36 .Similarly, a previous study reported that UGT1A1 SNPs rs10929303, rs1042640 and rs8330 are associated with acetaminophen glucuronidation activity 34 .Another study found that UGT1A1 SNP rs8330 is associated with the onset and prognosis of non-small cell lung cancer 37 .Our study reveals that UGT1A1 polymorphisms at SNPs rs10929303, rs1042640 and rs8330 are associated with the status of exposure to Agent Orange/Dioxin.Furthermore, SNPs rs1092030 and rs8330 have been associated with different enzymatic activities, resulting in distinct glucuronidation phenotypes, including fast, intermediate, and slow metabolism.Specifically, individuals carrying the rs1092030CC and rs8330CC genotypes exhibit a fast glucuronidation phenotype, those with the rs1092030CT and rs8330GT genotypes display an intermediate glucuronidation phenotype, and individuals with the rs1092030TT or rs8330GG genotype demonstrate a slow glucuronidation phenotype 11 .We observed that individuals carrying the rs1092030CC, rs1042640CC and rs8330CC genotypes exhibited the lowest levels of 2,3,7,8-TCDD, indicating a fast glucuronidation phenotype.Conversely, those carrying the rs1092030CT, rs1092030TT, rs1042640CG, rs1042640GG, rs8330CG, and rs8330GG genotypes had higher levels of 2,3,7,8-TCDD, representing slower glucuronidation phenotypes.These findings suggest that UGT1A1 gene polymorphisms (rs10929303, rs1042640 and rs8330) are factors significantly contributing to the metabolism of 2,3,7,8-TCDD.
Gene variation may influence gene expression, resulting in changes in the function and composition of essential pathway components, thereby influencing disease progression 38 .In particular, variants in the promoter and coding regions of UGT1A1 can affect UGT1A mRNA expression levels and UGT1A1 protein synthesis 17 .A variant TATAA element in UGT1A1 frequently causes decreased UGT1A1 expression and enzyme activity, increasing the risk of clinical outcomes such as jaundice, present in both types 1 and 2 of Crigler-Najjar syndrome 39 .UGT1A1*28 variant is related to irinotecan-induced toxicities in Caucasian patients with colorectal cancer 40 .The UGT1A1 SNP rs8330 is associated with gene expression, individuals with genotype CC exhibiting decreased expression of the UGT1A1 gene compared to patients with genotypes GG and CG 37 .Consistent with previous observations, our findings indicate that UGT1A1 SNPs rs10929303, rs1042640, and rs8330 are associated with increased levels of UGT1A1 gene expression in individuals exposed to Agent Orange/Dioxin but not in controls.Therefore, UGT1A1 gene polymorphisms (rs10929303, rs1042640, and rs8330) may be related to the metabolism of 2,3,7,8-TCDD, possibly by regulating UGT1A1 gene expression.However, further studies are needed to clarify this association and the role of UGT1A1 enzymatic activity in 2,3,7,8-TCDD metabolism.
Variants in the UGT1A1 exon or promoter regions can cause structural or functional defects in the protein, resulting in a decline in enzyme activity.Several studies have reported that SNPs in the UGT1A1 gene can regulate UGT1A1 enzyme activity 24,41,42 .Additionally, UGT1A1 gene variants can lead to a deficiency of UGT1A1 enzyme activity, as observed in Gilbert syndrome (GS) and Crigler-Najjar syndrome (CNS) 39 .Similarly, we found that UGT1A1 SNP rs10929303 was related to increased UGT1A1 enzyme concentrations in the group exposed to 2,3,7,8-TCDD.
Our results indicate that exposure to dioxin had an effect on UGT1A1 gene expression, leading to an elevation of the UGT1A1 enzyme concentration in dioxin-exposed groups compared to healthy controls.Particularly, dioxin exposure may promote UGT1A1 mRNA expression and/or enzyme degradation while inhibiting protein synthesis and/or enzyme activity.Earlier studies also observed that 2,3,7,8-TCDD can induce UGT1A1 gene expression 19,43 and that Agent Orange/Dioxin exposure can inhibit the activity of neuronal acetylcholinesterase (AChE) in an in vitro study 44 and pyruvate kinase in a clinical observation 26 .Therefore, our findings suggest that UGT1A1 gene polymorphisms (rs10929303, rs1042640, and rs8330) as well as exposure to 2,3,7,8-TCDD contribute to the regulation of UGT1A1 gene expression.
Although this study is the first to examine UGT1A1 gene polymorphism, gene expression, and enzyme concentration in individuals exposed to Agent Orange/Dioxin in Vietnam, it has several limitations.The first limitation is the lack of 2,3,7,8-TCDD concentration data in the control group.However, we carefully selected individuals who had never been exposed to Agent Orange/Dioxin or had any metabolic conditions linked to the 1.0 -0.5 In conclusion, our findings indicate that Agent Orange/Dioxin exposure is associated with UGT1A1 gene polymorphisms, UGT1A gene expression levels, and UGT1A1 enzyme concentrations.Exposure to 2,3,7,8-TCDD may influence UGT1A gene expression and enzyme concentrations.Our results suggest that exposure to 2,3,7,8-TCDD may impact human health by regulating UGT1A1 gene expression and its functional activity.However, further studies are needed to clarify the role of UGT1A1 gene variation and expression in dioxin metabolism. https://doi.org/10.1038/s41598-024-54004-0www.nature.com/scientificreports/

3 , 7 , 8 - 0001 AFigure 1 .
Figure 1.UGT1A1 gene expression levels and its association with dioxin exposure.(A) UGT1A1 gene expression levels in individuals exposed to Agent Orange/Dioxin and in healthy controls.P values were calculated by using the Mann-Whitney U test.(B) The effect of 2,3,7,8-TCDD exposure on UGT1A1 mRNA expression levels was assessed by using Benchmark dose (BMD) model.

Figure 4 .
Figure 4. Association of UGT1A1 SNPs and haplotype with gene expression in dioxin group and control group.UGT1A1 mRNA expression levels in subgroups with different genotypes of the UGT1A1 SNPs rs10929303 (A), rs104260 (B) and rs8330 (C) and in subgroups with haplotype CCC and TGG based on three UGT1A1 SNPs rs10929303, rs1042640 and rs8330 in individuals exposed to Agent Orange/Dioxin and in healthy controls.P-values of 0.02, 0.037, 0.023 and 0.007 were calculated for A, B, C and D, respectively, using the Kruskal-Wallis test for Dioxin group.For healthy group, P-values were > 0.05.

Figure 5 .
Figure 5. Association of UGT1A1 SNPs and haplotype with UGT1A1 enzyme concentrations in the dioxin group and control group.Enzyme UGT1A1 concentrations in subgroups with different genotypes of the UGT1A1 SNPs rs10929303 (A), rs104260 (B) and rs8330 (C) and in subgroups with haplotype CCC and TGG based on three UGT1A1 SNPs rs10929303, rs1042640 and rs8330 in individuals exposed to Agent Orange/ Dioxin and in healthy controls.P-values of 0.02, 0.073, 0.073 and 0.294 were calculated for A, B, C and D, respectively, using the Kruskal-Wallis test for Dioxin group.For healthy group, P-values were > 0.05.

Figure 6 .
Figure 6.Correlations of UGT1A1 gene expression levels with UGT1A1 enzyme concentrations in the dioxin group and control group.(A) The correlation between UGT1A1 gene expression levels and UGT1A1 enzyme concentrations in individuals exposed to Agent Orange/Dioxin.The selected model is Expon.m5 (AICs of the Expon.m5 model is 26.58 vs. 36.14 of the null model).(B) The correlation between UGT1A1 gene expression levels and UGT1A1 enzyme concentrations in healthy controls.The selected model is Expon.m3 (AICs of the Expon.m3 model is − 2.14 vs. − 0.28 of the null model).

Table 1 .
Genotype and allele distribution of UGT1A1 gene polymorphisms in study groups.P values were calculated by using Chi-square tests.Bold ORs and P values indicate statistical significance.